1. Field of the Invention
The instant invention relates to the fields of molecular biology, diagnostics and therapeutics. More particularly, the invention relates an isolated nucleic acid sequence encoding a novel endogenous precursor for granzyme B in non-immune cells (GrB-NIC). The invention also relates to methods of detection, expression, or inhibition of GrB-NIC in non-immune cells to modulate apoptosis and maintain tissue homeostasis for prevention and therapy of human diseases.
2. Description of Related Art
Human granzyme B (GrB) is a member of a subfamily of serine proteases originally found in granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Since its cloning in the late 1980s (Lobe et al., 1986; Schmid and Weissmann, 1987; Trapani et al., 1988), GrB has been mainly, if not exclusively, implicated in immune cell-mediated target cell killing (Yang et al., 1998; Yasukawa et al., 2000). The critical role of GrB in DNA fragmentation and apoptosis of target cells was established by compelling studies using CTL from GrB-deficient mice (Heusel et al., 1994). In a wide range of cellular immune reactions against bacterium/virus-infected, alloreactive, or neoplastic cells, GrB is produced by activated cytotoxic lymphocytes and stored in cytoplasmic granules; after effector-target cell conjugation, these granules are exocytosed, releasing GrB and other cytolytic proteins including a pore-forming protein (perforin). Then, perforin-dependent and perforin-independent mechanisms, the latter involving, at least in part, an insulin-like growth factor-II receptor (IGF-IIR) on the target cell surface (Shi et al., 1997; Motyka et al., 2000), facilitate the entry and intracellular trafficking of GrB in target cells. Once in the target cell cytosol, GrB leads to rapid induction of DNA fragmentation and apoptosis by activating downstream caspases (Yang et al., 1998) or through a Bcl-2-inhibitable mitochondrial pathway (Pinkoski et al., 2001).
Although generally associated with cytotoxic lymphocytes, more recently, albeit controversially (Graubert et al., 1997), expression of GrB-like protease was reported in other normal and malignant hematopoietic (non-lymphoid) cells, such as pluripotent stem cells capable of giving rise to all hematopoietic lineages, mobilized CD34+ hematopoietic progenitor cells, acute myeloblastic leukemic cells under genotoxic stress (Berthou et al., 1995; Bruno et al., 2000), epidermal keratinocytes (Berthou et al., 1997), testis and placenta (Hirst et al., 2001). In these later studies, however, expression of so called “GrB” mRNA was demonstrated only by in situ hybridization using antisense GrB RNA probes. The method was not able to determine the extent of the nucleotide sequence identity between more or less closely related RNA species, nor the size of the transcripts. Independent studies reported by others failed to detect GrB mRNA expression in mobilized hematopoietic CD34+ progenitor cells when an S1 nuclease protection assay was employed.